Unit 6 Planar Chromatography 5x641b

UNIT 6 PLANAR CHROMATOGRAPHY

Planar Chromatography

Structure 6.1

Introduction Objectives

6.2

Paper Chromatography Principle Stationary Solvent Systems Development of Chromatogram Detection Methods Applications

6.3

Thin Layer Chromatography Stationary Phases Mobile Phases Apparatus and Requirements Detections Methods Plate Concept Applied to TLC High-Performance Thin Layer Chromatography (HPTLC) Applications

6.4 6.5 6.6 6.7 6.8

6.1

Quantitative Aspects of PC and TLC Comparison of PC and TLC Summary Terminal Questions Answers

INTRODUCTION

So far you have studied about the general principles of chromatography where theoretical principles including resolution and plate concept were dealt. You now know that chromatography is now a very powerful separation technique used not only for the separation of complex mixtures but it is also used for quantification of each constituent. You have also learnt about classification of various chromatographic techniques which include a wide range of ion-exchange chromatography, affinity chromatography, gel filtration, electro chromatography, zone electrophoresis, size exclusion chromatography etc. In this unit, you will learn about planar chromatography, which includes paper chromatography (PC), thin layer chromatography (TLC) and electro chromatography (EC). Each of these techniques make use of a stationary phase in the form of a sheet or flat surface of a paper or any other material such as metal, glass or plastic plate coated with suitable adsorbent with the help of a binder. The mobile phase moves through the stationary phase by capillary action, assisted by gravity or an electrical potential. Here, we shall discuss only two techniques i.e. PC and TLC and a comparative study of their principles, methodology and applications will be undertaken. Once upon a time, term planar chromatography included two-dimensional chromatography though it has now come to signify the coupling of two chromatographic techniques with different mechanisms of separation. It is called ‘planar’ because the stationary consists of the plane surface of a paper or smooth glass plate. It includes paper chromatography (PC) and thin layer chromatography (TLC) which are the simplest of all other forms of chromatographic techniques. It also includes electrophoresis or electro chromatography where the movement of mobile phase is assisted by electrical potential. However, it will not be discussed here. The paper chromatography (PC) and thin layer chromatography (TLC) have the advantage of being simple, fast and inexpensive. These have been widely used for the 53

Chromatographic Methods-I

qualitative identification of different constituents in a mixture though these could be also used for quantitative determination of the components. However, PC is not used so commonly these days and as of now, planar chromatography based on TLC has found widest applications in organic synthesis laboratory, drug industry, clinical research and for investigating biochemical processes. In both the cases, sample is spotted with a micropipette on to a paper or a plate and then the chromatogram is developed using a suitable organic solvent. Each constituent present in the sample is identified on the basis of colour development using a suitable detection system. Different components of a solute travel with different speeds and the distance travelled by each component with respect to that of solvent gives a parameter called ‘retardation factor’ or Rf. Even though PC and TLC have many things in common but there are some basic differences in their operation. Here, we shall discuss the basic principles, apparatus required, methodology and some typical applications of both the techniques in the separation of complex mixtures of organic and inorganic compounds. We shall first describe paper chromatography, its principle, methodology and applications. PC is the simplest of all the chromatographic techniques but it is not so widely used these days. Later, we shall discuss thin layer chromatography, its methodology and applications including modern developments of plate concept and high-performance thin layer chromatography. that though PC and TLC remain primarily qualitative techniques but these could also be used for quantitative analysis. A comparative study of the two techniques will also be presented

Objectives After studying this Unit, you should be able to

54

•

explain the meaning of planar chromatography,

•

describe the principle of paper chromatography (PC),

•

give the meaning of Rf and factors affecting it,

•

discuss the type of paper used as and the types of solvent mixtures used in paper chromatography,

•

describe how to run and develop the paper chromatogram,

•

explain the methodology of separation of inorganic and organic mixtures,

•

give the potential applications of paper chromatography,

•

describe the principle of thin layer chromatography (TLC),

•

list the types of s and the types of mobile phases used in TLC,

•

explain how to run and develop the thin layer chromatogram,

•

discuss the advantages of two-dimensional paper and thin layer chromatography,

•

apply plate concept to TLC,

•

describe the basic principle of high-performance TLC (HPTLC),

•

discuss the potential applications of TLC,

•

describe the quantitative aspects of PC and TLC; and

•

compare PC and TLC.

6.2

Planar Chromatography

PAPER CHROMATOGRAPHY

It is one of the oldest and simplest techniques for qualitative analysis though it can also be used for quantitative determination. During later part of nineteenth century Runge, Schnbein and Goppelsroeder separated coloured dyes and other chemicals on paper or cloth which is considered as old cousin of paper chromatography. In 1944, R. Consden and A. H. Goron, two coworkers of A. J. P. Martin, the Nobel Laureate, first reported the separation of a mixture of amino acids and laid the foundation of paper chromatography. The technique is based on the movement of solvent phase in upward or downward direction by gravity and accordingly, it can be categorized as ascending or descending PC. There is another version of it, called circular paper chromatography where a circular paper is taken instead of strip and the solvent phase moves in circular direction. However, it is not very commonly used.

6.2.1 Principle The technique of paper chromatography consists of a sheet of cellulose filter paper which serves as a stationary phase or separation medium. A small amount (usually a few micrograms) of solute is placed in a small area near the end of strip. A solvent is allowed to move from the end of the paper by capillary action and after equilibration for some fixed period, the solute migrates from its initial point of application. The components of mixture are separated completely or partially in distinct coloured zones or are located by the application of different reagents or by applying ultra-violet fluorescence. At first, PC was considered as simply a form of liquid-liquid partition. The hydrophilic fibers of paper can hold (or bind) water in humid atmosphere such that a large percentage of water, say > 20% by weight, may be held in filter paper. Thus, paper was considered to be the analog of a column containing a stationary aqueous phase whence solute molecules get partitioned between this water and the mobile immiscible organic solvent. Later, this model was considered to be too simple because separations were also obtained where mobile phase was miscible with water or in other cases where it was just aqueous phase. Thus, it cannot be considered as simply liquid-liquid partition mechanism. Instead, besides adsorption and hydrogen bonding, interactions between solutes and the cellulose are involved. During the pulping and bleaching operations of paper, carboxylate and other ionizable groups are introduced into cellulose which makes the paper as ion exchanger. Rf value: It is a characteristic parameter called retardation factor and abbreviated as Rf. It represents the position of an ion or a substance with respect to solvent phase. Rf of a solute is defined as the ratio of the rate of movement of the solute to the rate of movement of the solvent. Rf is most commonly used in paper and thin layer chromatography and is considered as a characteristic of nature of the solute sample which may, of course, change with the solvent phase. It describes relative migration of the solute with respect to the solvent and may be represented as

Rf =

Distance traveled by the solute Distance traveled by the solvent

=

ds dm

… (6.1)

where, ds and dm are linear distances measured from the line of origin where spots are put as illustrated in Fig. 6.1. By definition, Rf value cannot exceed 1.0. Ideally, Rf values must be in the range of 0.1 to 0.9 with a minimum separation of 0.05. In order to have better separation, two spots must not overlap with each other and these must be symmetric without any tailing. In order to avoid tailing, different solvent mixtures, mixed in proper ratio, must be tried. If the spot of the solute is not symmetric, then ds is measured from the position of maximum intensity or the centre of the spot.

55

Chromatographic Methods-I

Fig. 6.1: Procedure for calculation of Rf value in paper chromatography

It has been observed that Rf values are influenced by the impurities in the paper and solvent, temperature and saturation of the atmosphere. Following factors may be considered; i)

Presence of other ions e.g. presence of chloride is carried out with nitrate solutions.

ii)

Acidity of the original solution-This may be needed to avoid hydrolysis and its need in the formation of soluble complex.

iii)

Development time- Sometimes it increases with the running time. Therefore optimum time may be determined.

iv)

Presence of other cations or anions as impurities.

v)

Ambient temperature.

Since Rf value depends on the distribution of complex species of the cations with different organic solvents, solvents should be carefully chosen. The technique of paper chromatography is primarily used for qualitative identification though it could also be used for quantitative determination but with a poor precision. Hence, it could be at best considered as semi-quantitative technique.

SAQ 1 Which of the following phenomenon is responsible for the rise of solvent in paper chromatography? i)

Capillary action

ii)

Gravity

iii)

Ion-exchange

iv)

Chemical affinity

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………...

SAQ 2 What is the ideal range of Rf values? i) 0.0 to 0.9

ii) 0.5 to 1.0

iii) 0.05 to 0.95

iv) 0.1 to 0.9

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………...

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6.2.2 Stationary As mentioned already, the stationary material is a highly purified cellulose filter paper such as Whatman No. 1, 2, 31 and 3 MM or acetyl acid paper having hydrophilic affinity for water. The solvent penetrates the fiber and causes swelling of paper changing its dimensions. Polymeric cellulose structure contains several thousand anhydroglucose units linked through oxygen atoms. Alternatively, modified forms of paper such as impregnated with alumina, silica gel, hydrous zirconium oxide, ion exchange resin may be used. Sometimes paper is coated with chelating agent solution such as dimethylglyoxime, 8-hydroxyquinoline etc. However, it should not have any impurities of Ca2+, Mg2+, Fe3+, Cu2+ etc so as to avoid interference. The paper is impregnated either neat or dissolved in a volatile solvent. The solvent should evaporate slowly so that stationary phase may distribute homogeneously. In this case, coated liquid phase may interfere with the detection of separated spots. The paper shows following properties: 1.

weak ion-exchange properties

2.

adsorptive properties

3.

water holding property

4.

mild reducing agent

Planar Chromatography

The quality of paper and its porosity play an important role in paper chromatography as it determines the rate of movement of the solvent used. Thick paper with increased sample capacity may be used for preparative studies. The most suitable cardboards are Schleicher and Schull 2070, SS2171 which can take a load up to 1 g at each point of application. Generally, a paper strip is cut into 4 cm × 30 cm for one dimensional PC. For two dimensional PC, however, a 30 cm × 30 cm square sheet is commonly used.

SAQ 3 Explain why following types of paper can not be used for paper chromatography? i)

Ordinary filter paper

…………………………………………………………………....................................... …………………………………………………………………....................................... …………………………………………………………………………………………... …………………………………………………………………....................................... ii)

Glazed paper

…………………………………………………………………………………………... …………………………………………………………………....................................... …………………………………………………………………....................................... iii)

Butter paper

…………………………………………………………………………………………... …………………………………………………………………....................................... …………………………………………………………………....................................... iv)

Bond paper

…………………………………………………………………………………………... …………………………………………………………………....................................... ………………………………………………………………….......................................

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Chromatographic Methods-I

6.2.3 Solvent Systems The nature of solvent plays an important role in the development of paper chromatogram. The solvent should be free from impurities and dried before use. Polar phase such as water is adsorbed by the paper and held stationary whereas the less polar solvent such as ethanol, acetone, glycol, formamide, acids, and amines flow through easily. Though pure solvent may be used but a mixture of solvents is preferred. Many solvent mixtures can be used provided these are not immiscible with one another. The following criteria may be adopted for the choice of the solvent; •

The solvent should not react chemically with any of the components of the sample mixture.

•

The composition of solvent mixture should not change with time. It means that none of its components should be volatile.

•

The solvent should not interfere with the detection of spots.

•

The distribution ratio should be independent of solute concentration.

•

The minimum difference between the Rf values of any two components should be 0.05 or 0.1 so that they may be separated easily.

Some of the solvents commonly employed for the separation of cations are mentioned below; Typical Mobile Phases for P C

Isopropanol,-ammonia -water (9:1:2) n-Butanol-acetic acid -water (4:1:5) Water-phenol Formamide-chloroform Formamide-chloroform -benzene Formamide-benzene Formamide-benzene -cyclohexane Dimethylformamide -cyclohexane Kerosene-(7:3)-isopropanol Paraffin oil-dimethylform amide -methanol-water

i)

n-Butanol saturated with 3M hydrochloric acid: equal volumes of alcohol and acid are shaken together and the upper layer is used.

ii)

Acetylacetone saturated with water: 7.5 mL acetylacetone is mixed with 0.05 mL hydrochloric acid and 2.5 mL dried acetone.

iii)

Acetone containing 5% (v/v) water and 8% (v/v) hydrochloric acid

iv)

Glacial acetic acid containing 25% (v/v) dried methanol

v)

Methanol

vi)

Methyl ethyl ketone containing 30% (v/v) water and 1% (w/v) potassium thiocyanate

vii)

Methyl acetate containing 3% (v/v) methanol and 10% (v/v) water

viii) Pyridine containing 10% (v/v) water ix)

Dried n-butanol containing 40% (v/v) dry methanol

The solvents must be refluxed over suitable drying agent such as potassium hydroxide for acetone and ethyl methyl ketone, or anhydrous calcium sulphate for n-butanol or as prescribed in literature.

6.2.4 Development of Chromatogram Paper strips are cut in appropriate size (usually 4-5 cm × 35-40 cm for a single spot but breadth may be changed for multiple spots) and stored under controlled conditions of humidity. A thin pencil line is drawn across the paper 2-3 cm from the edge and a circle is put at its centre. The sample is dissolved in a volatile solvent and it is spotted in the centre of line using a lambda (or micro) pipette of usually 5-10 micro liters or even with a capillary in case of qualitative analysis. The spot must be as small as possible without any spread for better separation and symmetric spots offer separation. It is best done by placing the sample drop wise, drying it with hot air blower (or hair drier) to evaporate the solvent as illustrated in Fig. 6.2. Sample spot may also be dried using infrafil lamp in a chamber. After drying, another drop may be put and dried. Several spots of different samples or a sample and the standard can be made across the line with a minimum separation of 1- 2 cm. The paper is now ready for development.

58

Planar Chromatography

Fig. 6.2: Illustration of (a) spotting of solute sample on paper using a capillary tube and (b) drying process using hair dryer

Important equipment used in paper chromatography is a development chamber where chromatogram is developed in a controlled environment. Some commercially available development chambers used in ascending and descending PC are shown in Fig. 6.3. In ascending chromatography, the paper is ed by means of a clip as shown in Fig. 6.3 (a).

Fig. 6.3: Some typical Developing chambers for paper chromatography: (a) Ascending and (b) Descending

In descending PC, the top edge of the paper is held down by a glass rod or strip as shown in Fig. 6.3 (b). The development chamber is presaturated with the solvent

59

Chromatographic Methods-I

system and then paper is hung with its end dipping in the solvent system as shown in the figure. The solvent starts rising slowly and then it stops after some time. It may take an hour or even longer. The development time will depend on the complexity of the mixture of solutes being separated, solvent system and the quality of paper and the ambient temperature. The diffusion of the solvent and the resulting separation into spots is termed as development of the chromatogram. It is essential that paper should be equilibrated with the solvent vapours in the chamber. For good resolution, reasonable Rf values must be in the range of 0.4 to 0.8 with typical separation time being 1 to 2 hours. Following are the main sources of error: •

Lateral diffusion of the solutes.

•

Variation in the structure of paper.

•

These become important especially for the concentrated solutes.

•

Variation in the geometry of the assembly for the standard and unknown samples. However, this error can be easily eliminated by using the same tank for the two samples where similar experimental conditions are maintained.

6.2.5 Detection Methods After development of chromatogram, the solvent front is marked and the solvent is dried. The spots of the separated compounds are then detected in a variety of ways by using any one of the following methods. i)

Reactions with colouring reagents such as dimethylglyoxime for Ni and hydrogen sulphide gas for any of Gr II elements

ii)

Fluorescence producing reagents

iii)

Inherent visible colours of the components such as sulphides and oxides

iv)

Radioactivity measurement of radiotracers

v)

Electrochemical methods such as potentiometry, conductance measurements and polarography have been successfully used for the detection and quantitative analysis in paper chromatography. However, these methods are of limited importance and not commonly used.

The reagents employed include diphenylthiocarbazone (dithizone), rubeanic acid, diphenyl dithiocarbazide, alizarin, salicylaldoxime, morin, potassium ferrocyanide, potassium chromate, ammonium sulphide and hydrogen sulphide gas. In many cases, two or more of these reagents are advantageous. The spots are characterized by their characteristic Rf values. Identical Rf values for a known and an unknown compound using several different solvent systems provide a good evidence that two are same especially if they run side by side along the same strip of paper. Example

60

i)

In a paper chromatographic separation of cations- Ag+, Pb+ and Hg+, solvent front rises to 18.4 cm while cationic spots were observed at 15.8, 12.1 and 5.9 cm, respectively. Calculate Rf values of the metal ions.

ii)

Emission gases of a two wheeler were tested for pollutant metals by paper chromatography. A spot corresponding to Rf value of 0.65 was observed. What is the possible pollutant metal ion in the emission gases?

Solution:

Here,

Rf value for Ag+ = 15.8/18.4 = 0.86 +

Rf value for Pb = 12.1/18.4 = 0.66

Planar Chromatography

Rf value for Hg+ = 5.9 /18.4 = 0.32 As Rf value of 0.65 is comparable to 0.66 corresponding to Pb+ , it can be concluded that the pollutant in the emission gases is likely to be lead.

6.2.6 Applications Paper chromatography has been widely used for separation and identification of cations in inorganic mixtures, organic functional groups, proteins and enzymes in biochemical work. It is particularly useful in the separation of closely related compounds such as isomers, homologues, isotopes and species having different valency or oxidation states. It has been found especially useful in the following: •

identification of trace metals in ores,

•

checking purity of pharmaceuticals and fermentation,

•

ripening of fruits and fermentation products,

•

detection of adulterants and contaminants in foods and drinks by comparing chromatogram of pure compound with that of the adulterant.

Sometimes, it happens that all the components of a mixture can not be separated using a single solvent system; some components separate better in one solvent and some in another. In such cases, two-dimensional paper chromatography may be employed. In this technique, a square paper of 30 cm x 30 cm is taken and the sample is spotted at one corner of the sheet. Chromatogram is developed using one solvent whence solute migrates parallel to one edge of the paper. Next time, the paper is turned at 90o and developed in a second solvent system which carries the solutes into the unused portion of the paper. A typical chromatogram of a protein hydrolysate using ninhydrin-stained spots is shown in Fig. 6.4.

Fig. 6.4: Illustration of two-dimensional paper chromatogram of a protein hydrolysate where solute is placed in corner at H and it is first run towards the right with an acetic acid-butanol solvent and then at perpendicular using phenol + resol/water solvent

Some typical applications are described below:

61

Chromatographic Methods-I

i)

Separation and Identification of Mn, Ni, Co and Zn A combination of Mn, Ni, Co and Zn may be separated by paper chromatography using Whatman No. 1 filter paper strip by developing with a mixture of acetone, water-hydrochloric acid system. The spots are located by the colouring reagents. These elements are identified by comparing with their characteristic Rf values of known with those in unknown mixture. The characteristic Rf values are as follows: Ni Mn Co Cu

ii)

- 0.09 - 0.21 - 0.43 - 0.61

Separation of a mixture of pesticides A combination of pesticides such as aldrin, endrin, lindane, heptachlor, DDT, BHC etc. may be separated on Whatman No. 1 filter paper treated with refined mineral oil in diethyl ether (5% v/v) and washed with 75% aqueous acetone. The mobile phase used is 3:1 methanol-water (v/v). The Rf values for aldrin, heptachlor, DDT, endrin and lindane were found to be 0.37, 0.48, 0.60, 0.62 and 0.89, respectively.

iii)

Speciation of Different Anions of Sulphur In its unique application, paper chromatography has been successfully used for speciation studies of sulphur such as S2-, SO32-, SO42-,S2O32-, S4O62- using radiotracer 35S. Whatman No. 1 filter paper was used along with solvent mixtures of dioxane, n-butyl alcohol-1N ammonia (1:1:1) and acetone-isopropanol-liquor ammonia (1:1:1). Different spots corresponding to various anions can be identified by radioactivity measurements using a G. M. counter. The Rf values for SO42-, S2O32-, S4O62- and S2- were found to be 0.12, 0.43, 0.54 and 0.77, respectively. Similarly, Rf values for S2O32- and SO32- were found to be 0.14 and 0.61, respectively. Paper treated with silicone or paraffin oil permits reversed phase paper chromatography where mobile phase is a highly polar solvent. This technique is referred to as ‘reverse-phase PC’. Commercially available papers coated with adsorbent or ion-exchange resin offer additional applications of adsorption and ion-exchange paper chromatography.

iv)

Autoradiography using Radiotracers In this technique, a radiotracer or a radiolabelled solute sample is used. After development of chromatogram, it is kept in with a photographic film of the same size. After some time when the film is developed then dark spots are observed in place of spots corresponding to the movement of solute components. This is called autoradiography. It has been found to be especially useful for the study of distribution and metabolism of compounds istered to the plants or animals. In a unique experiment, the Nobel Laureate Calvin and coworkers identified intermediate steps involved in the photosynthesis of carbohydrates from atmospheric CO2 in the presence of light and chlorophyll by using 14C, 32P and 3H. Plants were placed in the atmosphere containing 14C labeled CO2 and irradiated with light as shown in Fig. 6.5. The plants were removed after different irradiation periods of light and the molecular components were separated using paper chromatography. The presence of radioactive atoms in a compound by following autoradiography was taken as a proof of the involvement of that compound in the photosynthesis.

62

Planar Chromatography

Fig. 6.5: Illustration of Autoradiography method used for the determination of compounds involved in photosynthesis

6.3

THIN LAYER CHROMATOGRAPHY

Thin layer chromatography is different from paper chromatography in that a flat 63

Chromatographic Methods-I

surface of glass, metal or plastic coated with adsorbent such as silica gel, alumina or any other material, is used. The technique discovered by Ismailoff and Scraiber in 1938 is faster, more sensitive and has better resolution than paper chromatography. TLC is often used to develop optimal conditions for separation by liquid column chromatography. The technique has become a workhorse of the drug industry for purification of products. It has also found widespread use in clinical, industrial and environmental laboratories. Recent developments have elevated it from the level of semi-quantitative analytical procedure to one in which highly reliable quantitative separations can be performed.

6.3.1 Stationary Phases The stationary phase used in TLC can be an adsorbent, an ion exchanger, a molecular sieve or it can serve as the for a liquid film. It consists of a finely divided powder of particle size 5 to 50 µm. A variety of stationary s are available for coating the smooth surface of glass, metal or plastic. Plastic sheets have the advantage that these can be easily cut to any shape or size as required l. Silica gel G (60-120 mesh) remains the most frequently used coating materia. It contains hydroxyl groups on the surface which form hydrogen bonds with polar molecules. The adsorbed water prevents other polar molecules from reaching the surface. Hence, the gel is activated by heating to remove the adsorbed water. In modified silica gel, H and OH groups can be replaced by other functional groups similar to bonded phase. Alumina containing hydroxyl groups or oxygen atoms is another commonly used adsorbent. Other adsorbents widely used are powdered cellulose, magnesium silicate, calcium silicate, activated charcoal, natural diatomaceous earth, ion exchange resins such as Dowex50W-strong acid cation exchanger in sodium or hydrogen form and Dowex-1-strong base anion exchanger in chloride form. Kieselghur is often used for the separation of sugars. In fact, any adsorbent material that can be used in adsorption or column chromatography can be used in TLC. Here, an aqueous slurry of the powder is prepared by mixing the adsorbent with a binder such as plaster of Paris (CaSO4) and polyvinyl alcohol to help it adhere to the backing material. It is uniformly spread over the plate manually or by using one of the commercial forms of spreader as shown in Fig. 6.6. The thickness of the layer is usually in the range 0.1 to 0.3 mm but for preparative work much thicker layers are preferred. The solvent is evaporated off and adsorbents are activated by placing in an oven at 110o C for a few hours. Precoated ready-to-use plates and sheets are also commercially available.

Fig. 6.6: Apparatus used for preparing TLC plates where the applicator is filled with the slurry of adsorbent and binder in a solvent.

Thin layers of Sephadex superfine gel can be prepared for size exclusion. Gel is soaked in water for a few days and then spread on the plate. These are not dried but stored wet. Capillary action through these molecular sieves is much slower, typically of the order of 1 to 2 cm/hour. Hence, it takes longer period to develop these plates.

64

The following precautions must be followed while handling TLC plates: 1.

The surface of the TLC plates should not be touched. The plates should be handled carefully by holding at the edges so as to avoid any contamination due to sweat.

2.

The plates should be cleaned thoroughly so as to remove any extraneous material that might contaminate the adsorbent.

Planar Chromatography

SAQ 4 Explain why the flat surfaces of the following materials cannot be used as an inert for the coating of the adsorbent in TLC? i)

Plywood

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………... ii)

Asbestos sheet

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………... iii)

Card board

…………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………... iv)

Glass with granulated surface

…………………………………………………………………………………………... …………………………………………………………………………………………...

6.3.2 Mobile Phases The choice of the mobile phase is largely empirical but some general guidelines can be formulated. A mixture of organic solvents and water with the addition of acid, base, or complexing agent to optimize the solubility of the components of a mixture can be used. It may be emphasized that a large degree of trial and error is involved in the selection of the mobile phase. The polar solvents can themselves become strongly adsorbed; thus, producing an undesirable situation of partition system. The following criteria may be adopted. i)

The developing solvent must be of the highest purity because even trace amounts of impurities may yield irreproducible results.

ii)

Good separation of polar or ionic solutes can be achieved with a mixture of water and n-butanol. The criteria used for the selection of developing solvent are the same as for column chromatography.

iii)

The eluting power of solvents increases in the order of their polarities e.g. from hexane to acetone to alcohol to water. An eluotropic series, given in Table 6.1, can be used for the selection of the best solvent or the solvent mixture for a sample.

iv)

If the stationary phase is hydrophobic, mixtures of benzene, cyclohexane, and chloroform in different ratios provide satisfactory mobile phase. 65

Chromatographic Methods-I

Table 6.1: Eluotropic series of mobile phase Solvent n- Pentane Cyclohexane Carbon tetrachloride Toluene Chloroform Methylene chloride Tetrahydrofuran Acetone Methyl acetate Acetonitrile n- and iso-propanol Ethanol Methanol Water Ethylene glycol

Solvent strength εo 0.00 0.04 0.18 0.29 0.40 0.42 0.45 0.56 0.60 0.65 0.82 0.88 0.95 1.00 1.11

6.3.3 Apparatus and Requirements Commonly, the plates in sizes of 2.5×10, 2.5×15, 2.5×20, 5×20, 10×20, and 20×20 in centimeters are available. These are of two types: conventional and high-performance. The former have thicker layers of about 0.25 mm with particle size of < 20 µm. On the other hand, the high performance plates usually have thickness of 0.1 mm and particle size of <5 µm. As the name implies, high performance plates provide fast and sharp separations. In of plate theory, the conventional TLC will have about 2000 plates in 12 cm with development time of less than 30 minutes. Correspondingly, the number of plates in high-performance TLC will be about 4000 in 3 cm requiring 10 minutes development time. However, the high performance TLC has much less sample capacity. The sample application in TLC is the most critical aspect and particularly so in quantitative measurements. Usually the sample is dissolved in a suitable solvent with a concentration of less than 0.1% and applied as a spot of about 1 cm from the edge of the plate. The spot should be as small as possible and dried in a manner similar to PC, (Fig. 6.2). In case of dilute solutions, 3 to 5 drops are applied. The spotting may be done with a capillary tube or by use of a hypodermic syringe. However, mechanical dispensers with increased precision and accuracy are available commercially. After the sample solvent has evaporated, the plate is placed in a closed container presaturated with the developing solvent. Typical developing chambers used for ascending and horizontal thin layer chromatography are shown in Fig. 6.7.

Fig. 6.7: Typical developing chambers used for (a) ascending and (b) horizontal thin layer chromatography. In the later case, the sample is placed on both the sides of the plate and developed towards the centre

66

The plate is immersed in the developing solvent avoiding its direct . The developing solvent travels up the plate and after ing the point of sample application; it dissolves the sample and carries it up the plate. Thus, the sample distributes itself between the moving solvents and the stationary phase.

Planar Chromatography

After the developer has traveled about two-third of the length of the plate, it is removed from the container and dried. The positions of the components are then determined by any of the methods described in the next section.

6.3.4 Detection Methods The detection of the spots in TLC is easier than in PC as silica and alumina used as are inert than paper and hence, strongly reactive reagents can be used to locate the compounds. A universal technique involves the use of iodine vapours for colourless or non-fluorescent spots. It consists of exposing the developed plate to iodine vapours which interact with the sample components, either chemically or by solubility to produce colour. Alternatively some other colour forming reagents called chromogenic reagents as given in Table 6.2 may be used. Table 6.2: Some Chromogenic Reagents Used for Identification in Thin Layer Chormatography Reagent

Application

Ninhydrin or isotin 2,4-dinitophenylhydrazine H2S water, diphenylcarbazide Rubeanic acid Aniline phthalate Chloroplatinic acids Bromothymol blue Antimony trichloride

Amino acids Ketones and aldehydes Metals Metals Sugars Alkaloids Lipids Steroids, essential oils

Now-a-days, the thin layer plates and sheets incorporating fluorescent dyes in powdered adsorbent, are commercially available. When these are held under ultraviolet radiation, dark spots glow where sample spots occur due to fluorescent substances. Alternatively, an immobile fluorescent substance may be added while preparing the slurry so that the separated compounds appear as dark spots against fluorescing background when the plate is viewed under the ultraviolet light. Another common technique used for the identification of organic compounds is by spraying the plate with concentrated sulphuric acid solution and then heating it in an oven. Charring of the organic compounds make them appear as black spots. The Rf values in TLC are difficult to reproduce because of experimental variables. Following factors may be considered. i)

Nature of the adsorbent-its chemical nature, particle size, surface area and binder. Also its activity, thickness and uniformity.

ii)

Nature of mobile phase-its purity, moisture content, precision of mixing in case of mixture and volatility.

iii)

Amount of sample used

iv)

Vapour-pressure equilibrium between the plate and the atmosphere of the development chamber

v)

Ambient temperature 67

Chromatographic Methods-I

A typical thin layer chromatogram of a mixture is shown in Fig. 6.8.

Fig. 6.8: Thin layer chromatogram of a mixture

The chromatograms can be stored for future reference or their photographs can be made. Solved example Methanolic extract of a medicinal herb Terminalia Arjuna bark powder was ed through a silica gel column and eluted by various solvents. Petroleum ether-ethyl acetate (10:1) eluant spotted on TLC where three components A, B and C corresponding to Rf values of 0.63, 0.72 and 0.79, respectively were observed. One of these compounds was a carboxylic acid which was identified as tartaric acid. In order to further confirm, a known standard tartaric acid was run on TLC where solvent front ran up to 8.6 cm and its spot was found at 6.3 cm. Which one of the spots corresponds to tartaric acid? The Rf value for standard tartaric acid =

6.3/8.6 = 0.73

Thus, the standard value of 0.73 is comparable with 0.72 for B compound. Hence, B is tartaric acid.

6.3.5 Plate Concept Applied to TLC Plate concept and its as developed for column chromatography can be applied to thin layer chromatography after slight modifications. As described in sub-Sec. 6.2.1 and Eq. 6.1, the retardation factor Rf is given by the equation Rf = ds/ dm. In order to adapt, thin layer chromatography with the plate concept, a chromatogram may be visualized as a plot, shown in Fig 6.9, where the spot diameter may be considered as width in Eq. 6.6. Further, ds and dm may be defined in of retention time of solute (tr) and retention time of mobile phase (tm), respectively.

Fig. 6.9: Schematic representation of thin layer chromatogram with plot

68

For the mobile phase, tm may be defined as the distance divided by its linear velocity, u tm

=

ds /u

… (6.2)

Planar Chromatography

The solvent does not reach the same point until the mobile phase has travelled the distance dm. Therefore, tr

=

dm /u

Substituting the values of tr and tm in equation

… (6.3) tr = tm (1 + k′), we get

k′ = (dm – ds)/ ds

… (6.4)

The capacity factor k′ can also be expressed in of the retardation factor Rf as k′ =

(1 – ds/dm) / (ds/dm) = (1 – Rf)/ Rf

… (6.5)

This approach can be used for the method development in column chromatography. It is not easier to calculate capacity factor by thin layer chromatography but it is more rapid than obtaining it from column chromatography experiment. Also, this can be used for calculating the plate height by first determining the number of plates as follows: N = 16 (ds / w)2

… (6.6)

where w is the spot width as shown in Fig. 6.9. Thus, the plate height H is represented by H = dm /N

… (6.7)

6.3.6 High-Performance Thin Layer Chromatography (HPTLC) In order to have better resolution and sensitivity in TLC, it can be coupled with highperformance liquid chromatography (HPLC), mass spectrometry (MS), Fouriertransform infrared (FTIR) etc. As already mentioned in Sec. 6.3, TLC can be compared with liquid-liquid chromatography where solute is detected in the presence of the mobile phase which can be so chosen that it may not interfere with the detection wavelength. Reverse phase version of TLC has been proposed where better separation can be obtained than that with normal TLC. Thus, the performance of TLC can be improved by improving the quality of adsorbent and sample application procedure done by choosing smaller particle size, reducing the plate length and using smaller quantity of sample as described below. Quality of Adsorbent: Specially purified silica gel with average particle diameter of 3-5 µm is used and it may also be modified with chemically bonded layers. Such an adsorbent may have 5000 theoretical plates providing a much improved performance over the conventional TLC. Thus, a better separation may be achieved in much shorter time. Methodology of Sample Application: The amount of sample applied in HPTLC is much smaller, typical volumes being 100-200 nL with a compact diameter of 1.0-1.5 mm. Number of samples may be increased because of its compact size. After developing the plate up to a distance of 3 to 6 cm, compact separated spots are obtained which may have 10 times better detection limits than the conventional TLC. Application of the sample on the plate is a critical process in HPTLC. A convenient spotting device used is platinum-iridium capillary of 100-200 nL whose tip is polished to provide a smooth, planar surface of area 0.05 mm2. It is used with a mechanical applicator which minimizes damage to the plate surface. Quantitative analysis requires scrapping of the spot followed by dissolution or direct scanning. In order to shorten the procedure, solid phase extraction column is used to concentrate the sample, followed by automatic sampling device so that multiple samples may be processed in a short period and highly reproducible results are obtained. 69

Chromatographic Methods-I

Detection: In situ quantitative analysis is carried out by direct photometric measurements. For this purpose, single or double beam spectrophotometers are available. Scanning densitometers based on reflectance and absorbance of light are often used to measure the individual spots. The chromatogram is scanned with a moving beam of light and the intensity of reflected light from the plate surface is measured. The difference in light intensity between the thin layer plate without sample and with spot is displayed as peaks in the scan. The areas of peaks correspond to the amount of solute component in the sample. An alternate procedure consists in measuring the light transmitted through the plate. Photodensitometers measure the transmitted or reflected light to produce a photograph revealing dark and light zones for the areas of constituents in the sample. The standard deviation for quantitative determinations by densitometry is better than 5%. CAMAG, Switzerland have developed a HPTLC Vario system with key features of development with six different solvents side by side, sandwich as well as tank configuration making directly comparable results, and six different conditions of equilibration including relative humidity.

6.3.7 Applications Thin layer chromatography is widely used for qualitative analysis; almost any mixture can be partially resolved. Inorganic separation of metals in alloys, soils and geological samples and so also organic compounds formed during synthesis work or the analysis of natural products can be easily achieved by TLC. It is ideally suited for following the course of complex reactions, quality control, purity checks, plant extracts, biochemical preparations, clinical diagnosis, forensic tests etc. It is primarily used for qualitative identification by comparison of the Rf values with those of standards run under identical conditions or by removing the material from the chromatogram and subjecting it to further tests by other techniques. The versatility of this technique has resulted in a rapid spread of its use in all the fields. Particularly sharp separations have been obtained for vitamins, fatty acids, lipids in serum, amino acid s, dyes and glycerides, pesticides, sugars, etc. In a typical case, presence of gallic acid in trifala (a mixture of Harad, Baheda and Amla) was identified by thin layer chromatography in ethyl acetate-methanol (7:3) solvent system. The standard sample of gallic acid was spotted along side of unknown and compared as shown in Fig. 6.10. It is observed that the two samples give spots with Rf = 0.86. Presence of gallic acid was further confirmed by elemental analysis, infrared, NMR and GC-MS methods.

Fig. 6. 10: Identification of gallic acid in trifala using ethyl acetate – methanol (7:3) solvent system where comparable Rf values are observed

Similar to the two-dimensional paper chromatography described in sub-Sec. 6.2.5 and Fig. 6.4, the two-dimensional thin layer chromatography has been found to be very 70

effective for the separation of complex mixtures. The sample is spotted in one corner and developed with the first solvent. After thorough drying, the plate is turned through 90o and developed with the second solvent. The whole process of the development of chromatogram is shown in Fig. 6.11. It can be compared with a ‘standard map’ of known mixtures.

Planar Chromatography

Fig. 6.11: Schematic representation of two-dimensional thin layer chromatography; (a) sample is spotted in one corner, (b) results obtained after first separation, (c) rotation of plate by 90º and (d) final chromatogram

6.4

QUANTITATIVE ASPECTS OF PC AND TLC

As already emphasized, PC and TLC are primarily qualitative techniques used for identification of inorganic and organic compounds. However, both the techniques can also be used for quantitative analysis after taking proper care and suitable calibration so that reproducible and accurate results are obtained. Here, the most important aspect is that all the chromatographic conditions must be well standardized. The following precautions must be taken. i)

Standards and samples must be applied to the paper or plate in spots of similar size and at similar concentration using propipette.

ii)

All the solvents must be of high purity. While using mixtures, these should be prepared carefully.

iii)

Development chamber must be brought in equilibrium in the same manner.

After the development of chromatogram, the spot area must be removed carefully by cutting the paper or scratching out from the plate and measured. Relative precision of these measurements is usually of the order of 5-10% but can be 1-2%. The main difficulties in quantitative measurements lie in defining the boundaries of spots and controlling chromogenic reactions in a reproducible manner. Following are some of the physical and chemical methods employed. 1.

Visual comparison of spots: The standards containing known amounts and samples must be run on the same sheet or plate, and their relative intensity or area may be compared visually.

2.

Physical measurement of coloured spots: The colour intensity may be measured by transmission or reflectance using spectrophotometer. Scanning photo densitometers which measure spot intensity are also available. For organic fluorescent substances, the fluorescence intensity may be measured under illumination with ultraviolet radiation. Full spectrum recording and multiple wavelength scanning capabilities are available commercially. 71

Chromatographic Methods-I

3.

Spot area measurement: The spot area determined by using transparent graph paper and counting the squares within a spot, is proportional to the logarithm of the amount of substance. A standard is run under identical and controlled conditions and a calibration plot is made between area vs log of concentration.

4.

Radioactivity measurements: In this method, radiotracers or radiolabelled substances are used for spotting. The paper strip or plate after development can be scanned for activity. A comparison of the activity for the sample with that of standard may give quantitative results. Alternatively, before spotting pencil lines are drawn on the paper strip at an interval of one half cm and after development these strips are cut and then counted for radioactivity using GM counter or scintillation gamma ray spectrometer. Also, an X-ray film may be kept in with the developed paper or thin layer plate so that black spots will appear at the location of the separated constituents.

5.

Removal of spots: The spot is cut from the paper or removed from the plate by scrapping of the adsorbent. The substance is eluted or extracted from the spot paper or adsorbent (using dil hydrochloric acid and slight warming for inorganic substance and suitable organic solvent for organic substance). The solution is made up to a standard volume and then measurements are made by spectrophotometry or any other technique.

CAMAG, Switzerland, pioneers in modern equipments of TLC, have come up with a Planar Chromatography Manager winCATS which offers a new approach to thin layer chromatography. It is a 32bit Windows software designed to control, monitor and document all steps of TLC analysis. CAMAG instruments like the automatic TLC sampler, scanner etc. can be linked to winCATS by means of a software interface and offer unique features.

6.5

COMPARISON OF PC AND TLC

Even though PC and TLC both are considered as planar chromatographic techniques where a plain or flat surface is used and the main parameter is Rf . But, both these are widely different in of stationary , developing chamber, detection methods and even in applications. The PC has limited applications but the TLC has wide range of applications especially in drug industry and biochemical processes. For this reason, the PC technique has been superseded by TLC in analytical laboratories though it is still used for demonstrating the general principles of chromatographic separations. A comparison of TLC and PC separation of nucleotides is shown in Fig. 6.12

(a)

(b)

Fig. 6.12: A comparison of separation of nucleotides by TLC and PC under identical conditions: Solvent; saturated ammonium sulphate-1M sodium acetate-isopropanol (80:18:2) (a) Thin layer chromatogram using cellulose as adsorbent, Run time, 90 min(b) Paper chromatogram using Scheicher and Schull 2034 paper, Run time, 135 min

72

In the above figure one-dimensional development using identical conditions shows the superiority of cellulose layer TLC over PC for separating various mixtures. It may be observed that TLC shows reduced degree of spot diffusion or better resolution especially for samples 1 to 4.

Planar Chromatography

The following points of differences are of special significance: i)

In case of PC, cellulose paper acts as the stationary that is flexible whereas in TLC, suitable adsorbent material is coated on to a glass, metal or plastic sheet that is rigid.

ii)

In PC, the solvent rises by capillary action but in TLC solvent moves similar to that in liquid column chromatography.

iii)

The development of chromatogram in PC may take several hours but TLC is much faster as it takes only half an hour or so.

iv)

PC may be classified as ascending, descending or circular types though it is generally carried out in ascending mode. On the other hand, TLC is primarily ascending though it has a horizontal version as well.

v)

Developing chambers used in PC and TLC are somewhat different as the paper needs to be hung with a rod whereas the plate touches the bottom of the chamber in later case.

vi)

For quantitative analysis, the paper strip with solute spot needs to be cut with scissors whereas in TLC, the spot portion needs to scrapped. In both the cases, the solute is dissolved though paper is removed whereas adsorbent in TLC is filtered off.

vii)

PC takes more time and is less reproducible whereas TLC is much faster and more reproducible.

viii) PC and TLC both have been carried out using coated liquid phases and reversed phase versions have been developed. ix)

The plate concept has been adapted to TLC and high performance TLC has been developed whereas this is not the case with PC.

x)

The choice of paper in PC is limited whereas in TLC, a variety of stationary phase surfaces are available.

xi)

Not many automated instruments have been developed in PC contrary to TLC where automated sampler, capillary dispenser, developing chamber and documentation system with camera or software have been developed.

6.6

SUMMARY

In this unit, you have learnt about planar or two-dimensional chromatography which includes paper chromatography and thin layer chromatography. The main parameter in both the cases is retardation factor, Rf , which is defined as the relative movement of the solute components to that of the solvent phase. In both the cases, the details of stationary phase, mobile phase, detection methods and some typical applications are discussed. The plate concept has been adapted to TLC so that the separation process can be better understood. Modern developments in TLC include development of high performance TLC. A comparison of paper chromatography and thin layer chromatography is presented. Though both the techniques are primarily used for qualitative identification of inorganic and organic compounds but TLC is now being widely used for quantitative analysis. Use of radiotracers in both the techniques is also discussed.

73

Chromatographic Methods-I

6.7

TERMINAL QUESTIONS

1.

Spots 3 and 4 in Fig. 6.12 correspond to specific nucleotides 3′-GMP and 2′-GMP, respectively. Determine the Rf values in PC and TLC, compare and comment on them.

2.

A mixture of U, Mg and Al was separated by paper chromatography and then quantitatively determined by spectrophotometry. 100 ppm standards of each of these were prepared and reacted with oxine for U, magneson for Mg, aluminon for Al giving absorbance values 0.85, 0. 80, 0.65, respectively. Spot areas of paper chromatogram were cut, dissolved and their absorbances were found to be 0.39 for U, 0.42 for Mg and 0.53 for Al. Calculate concentrations of each of three elements in the mixture.

3.

i)

Explain the basic principle of Reverse Phase Chromatography and its applicability to PC and TLC.

ii)

Explain reverse phase PC and TLC. In what respect are these different from normal PC and TLC?

4.

Enumerate reasons for lack of modern developments in paper chromatography whereas in case of TLC, plate concept has been adapted and it has been further developed as HPTLC.

5.

i)

Explain the characteristics of paper used in PC.

ii)

Can van Deemter equation be applied to TLC?

iii)

Similar to iodine used for detection in TLC, can one use Br2 for the detection of organic compounds.

iv)

Explain how Rf value is different from retention time, tr. How do you justify the use of retention time in TLC?

6.

A six component mixture of compounds obtained by extracting plant leaves with an organic solvent was subjected to two-dimensional thin layer chromatography by placing a portion of the extract in one corner. First, it was developed with solvent A and after drying the plate and turning it to right angle, a different solvent was allowed to flow perpendicular to the direction of the first solvent. The black dot represents the start of original sample. Finally, the plate was sprayed with a reagent and the blue coloured spots were observed as shown in Figure.

7.

The Rf values of six components as reported for known compounds are listed in Table. Identify the numbered compounds in figure on the basis of their Rf 74

Planar Chromatography

values. Compound 1 2 3 4 5 6

6.8

Rf (solvent ) 0.0 0.8 0.5 0.8 0.2 0.8

Rf (solvent B) 0.5 0.0 0.5 0.8 0.4 0.5

ANSWERS

Self Assessment Questions 1.

i)

Capillary action

2.

ii)

0.1 to 0.9

3.

i)

Because of impurities

ii)

Solvent will not rise because of smooth surface

iii)

It has smooth surface on both sides.

iv)

It also has smooth surface.

i)

It has rough surface.

ii)

It has uneven surface.

iii)

Besides uneven surface, solvent will be absorbed by the cardboard.

iv)

It has uneven surface.

4.

Terminal Questions 1.

Measure Rf values in Fig. 6.12 and compare two values obtained from PC and TLC. Rf values obtained from TLC are likely to be more accurate than those obtained by PC.

2.

100 ppm standards of each of U, Mg and Al show absorbances of 0.85, 0.80 and 0.65, respectively. Considering absorbance for unknown mixture, concentration for different elements can be calculated as follows: Concentration of U = 0.39×100/0.85 = 45.9 ppm Concentration of Mg = 0.42×100/0.80 = 52.5 ppm Concentration of Al = 0. 53×100/0.65 = 81.5 ppm

3.

i)

In reverse phase chromatography applicable to liquid-liquid chromatography, stationary phase is non-polar or preferably long chain hydrocarbon and the solvent phase is polar such as chloroform, acetone, tetrahydrofuran or alcohols etc. Its applicability to PC and TLC are new developments where stationary phase is modified accordingly so as to obtain better separations.

ii)

In reverse phase paper chromatography, paper is coated with silicone or paraffin oil and the mobile phase is any polar organic solvent. Similarly, in case of TLC, adsorbent is modified accordingly and is coated on a smooth plate whereas the chromatogram is developed using a polar solvent. 75

Chromatographic Methods-I

4.

In case of PC, paper acting, as stationary phase is difficult to get in reproducible form. Further, it is difficult to get it in humid free form. Also, there is no universal detector in PC unlike in TLC where some common detectors like iodine and ninhydrin can be used. In addition, PC takes longer time for analysis compared to that for TLC. Only for these reasons very few attempts have been made with regard to automation in PC. Also, in case of PC, the particle size cannot be controlled whereas it is an essential condition in the conversion to high-performance form.

5.

6.

i)

Cellulose filter paper used in PC should be free from any impurities and humidity and it should not be wet.

ii)

No. van Deemter equation can not be applied to TLC.

iii)

Br2 cannot be used for the detection of organic compounds because it reacts with the double bonds present in the compounds and it is also hazardous to handle.

iv)

Rf and tr.values are different. By definition, tr = tM (1+k′) where k′ is the capacity factor representing column characteristics. However, Rf value is the ratio of distances travelled by the solute to that of the solvent.

In Figure, the top line from right to left, three spots correspond to compounds 2, 4 and 6, respectively. Second line from top, one spot corresponds to compound 3 Third line from top, one spot corresponds to compound 5 Bottom line, one spot corresponds to compound 1.

Further Readings

76

1.

Vogel’s Textbook of Quantitative Chemical Analysis by J. Menham, R.C. Denney, J.D. Barnes and M.J.K. Thomas, 6th Edn, Low Price Edition, Pearson Education Ltd, New Delhi (2000).

2.

Quantitative Analysis by R. A. Day and A. L. Underwood, 6th Edn, Prentice Hall of India, New Delhi (2001)

3.

Instrumental Analysis, Editors, H. H. Bauer, G. D. Christian and J. E. O’Reilly, 2nd Edn, Allyn and Bacon, Inc., Boston (1991)

4.

Principles of Instrumental Analysis by D. A. Skoog, F. J. Holler and T. A. Nieman, 5th Edn, Thomson Brooks/Cole, Bangalore (2004)

5.

Fundamentals of Analytical Chemistry by D. A. Skoog, D. M. West, F. J. Holler and S. L. Crouch, 8th Edn, Thomson Brooks/Cole, Bangalore, 2004,

6.

Analytical Chemistry by G. D. Christian, 6th Edn, John Wiley & Sons Inc, Singapore (2003)

7.

Principles and Practice of Analytical Chemistry by F.W. Fifield and D. Kealey, 5th Edn, Blackwell Science Ltd, New Delhi (2004)

8.

Handbook of Instrumental Techniques for Analytical Chemistry, Editor, F. Settle, Low Price Edition, Pearson Education Inc, New Delhi (2004)

9.

Instrumental Methods of Chemical Analysis by G. W. Ewing, 5th Edn, Mc-Graw Hill, Singapore (1985)

INDEX

Planar Chromatography

A. J. P. Martin 55 Activated alumina 41 Adsorbent type 41 Adsorption 6, 7, 8, 36 Affinity chromatography 6, 8 Applications of liquid column Applications Autoradiography 62 Calvin 62

Separation of pesticides 62 Speciation 62 Reverse-phase PC 62

Ascending chromatography 59 Auto radiography 62 Carrier gas velocity (u) 24 Chromatogram 14 Chromatographic columns 11 Chromatography 5, 6, 7, 49 Classification 7 Mechanism responsible for separation 7 Nature of mobile phase 7 Shape of the solid 7

Chromogenic reagents 67 Circular paper chromatography 55 Cross-linked dextrans 9 Dead time 16 Detection methods 60, 67 Chromogenic reagents 67 Dithizone 60

Development of chromatogram 58 Ascending chromatography 8, 59 Development chamber 59, 67

Development techniques 45 Dislacement development 45, 46 Displacer 46

Elution analysis 45, 46 Gradient elution analysis 47

Frontal analysis 45

Development time 56 Distribution coefficient 37 Distribution constant 15 Displacement development 46 Displacer 46 Dithizone 60 Durapck ® 42 Eddy diffusion 20 Eddy diffusion term (A) 21 Multiple flow path term 20, 21

Elution 13 Elution analysis 46 Equipment Column 40 Column packings 40 Detectors 40, 43, 48 Bulk property 40 General detectors 40 Selective detectors 40 Solute property 40

77

Chromatographic Methods-I

Mobile phase reservoir 39

Frontal analysis 45 Gas chromatography 7, 10, 13 Gas- liquid chromatography (GLC) 10 Gas- solid chromatography (GSC) 10

Gas-liquid chromatography 5 Gel filtration chromatography 6, 9 Gel permeation chromatography 9 Gradient elution analysis 47 Height equivalent to theoretical plate (HETP) 18 Henry’s law 23 High performance liquid chromatography 6, 9 High-performance thin layer chromatography HPTLC) 10, 69 CAMAG 70, 72 Detection 70 Platinum-iridium capillary 69

HPLC Basic aspects of 48

Ion exchange 5, 6 Ion exchange chromatography 5, 9 Amphoteric exchangers 9 Anion exchangers 9 Cation exchangers 9 Ion pair chromatography 9

Linear chromatography 15 Liquid chromatography 7, 8, 13, 20, 38 Adsorption phenomenon 36 Affinity chromatography 6, 8 Ascending chromatography 8 Descending chromatography 8 Extraction chromatography 8 Gel filtration chromatography 6, 9 Gel permeation chromatography 9 High performance or high pressure liquid chromatography 9 High pressure thin layer chromatography (HPTLC) 10 Ion exchange chromatography 9 Ion-pair chromatography 9 Liquid- liquid partition chromatography 8 Liquid- solid adsorption chromatography 8 Normal-phase chromatography 8 Reversed-phase chromatography 8 Size exclusion chromatography 9 Separation factor 27, 37

Liquid column chromatography 35, 38, 43 Applications 49 Experimental set up 38

Liquid-liquid partition chromatography 35 Liquid, gas and supercritical fluid Liquid-liquid chromatography 37 Capacity 37 Differential partitioning 37 Distribution coefficient 37 Partition coefficient K 37 Retention factor, k´ 37 Reversed phase partition chromatography 37

Liquid-liquid partition chromatography 8, 35, 43, 55 Liquid-solid adsorption chromatography 5, 8, 35 Liquid-solid chromatography 36 Liquid-solid column chromatography Durapak 42

78

Liquid-liquid partition chromatography 43 Stationary phases 42 Particle Size 42 Polar Adsorbents 41 Regeneration 42 Stationary phases 41, 42

Planar Chromatography

Activated alumina 41 Silica gel 41

Surface Area 41

Longitudinal diffusion 20, 22 Longitudinal diffusion term (B/u) 22 M. Tswett 5 Martin and Synge 5 Mass transfer between phases 20 Mass transfer term (CS) 24 Migration rates 15 Distribution constant 15 Linear chromatography 15 Non- linear chromatography 16

Mobile phase 6, 43 Retention factor 17

Retention time 16 Selectivity factor 18 Shapes of peaks 18

Mobile phase mass transfer coefficient (CM) 24 Nature of the mobile phase 7 Ninhydrin 67 Non-equilibrium in mass transfer term (Cu) 23 Henry’s law 23

Non-linear chromatography 16 Number of plates (N) 18 Optimum carrier gas velocity (Uopt.) 24 Paper chromatography (PC) 5, 7, 53, 55 Applications 61 Detection methods 60 Development of chromatogram 58 Principle 55 Solvent systems 58 Stationery phase 55 Stationery 57

Particle size 42 Partition 6, 10 Partition coefficient, K 37 PC and TLC Comparison 72 Physical measurement of coloured spots 71 Quantitative aspects 71 Radioactivity measurements 72 Removal of spots 72 Separation of nucleotides 72 Spot area measurements 72

Pesticides separation of 62

Physical adsorption 10 Planar chromatography 53 Plate concept 68 Plate count 18 Plate height (H) 18 Platinum-iridium capillary 69 Polar adsorbents 41

79

Chromatographic Methods-I

Polyacrylamide 9 Rate theory 20 Regeneration of adsorbent 42 Resolution 26 Retardation factor (Rf) 54, 55 Retention factor 17 Retention time 16 Dead time 16

Reverse phase PC 62 Reversed phase partition chromatography 37 Selectivity factor (separation factor) 18, 27 Separation factor 37 Separation medium 55 Shapes of peaks 18 Silica gel 41 Size exclusion 6 Size exclusion chromatography 9 Solvent strength 44 Solvent systems 58 Paper chromatogram 58 Solvents 56, 58, 65, 67, 68

Speciation 62 Stationary phase 6, 42, 55 Stationary phase mass transfer coefficient (CS) 24 Stationary 57 Supercritical fluid chromatography 6, 7, 11, 13 Critical point 11 Critical pressure 11 Critical temperature 11, 20 Supercritical fluid 11

Surface area 41 Theoretical plates 18, 20 Thin layer chromatography (TLC) 7, 53, 63, 68 Apparatus and requirements 66 Plates 66 Conventional 66 High-performance 66

Developing chambers 66

Applications 70 Gallic acid 70 Trifala 70

Detection methods 67 Chromogenic reagents 67

Mobile phase 65 Eluotropic series 66

Plate concept 68 Stationary Phases 64 Alumina 64 Sephadex 64 Slurry 64

Three-dimensional chromatography 7 Two-dimensional chromatography 7 van Deemter plot 24 van Deemter equation 20 Zone broadening 24

80